The long range goal of the proposed work is to define the molecular components and mechanisms mediating vibrio cholerae colonization to the point where there is sufficient knowledge to intelligently incorporate the participating molecules and any antigenic variants into the design of improved cholera vaccines. Most of this proposal involves a genetic analysis of TCP (toxin regulated pilus) mediated colonization. This includes oligonucleotide directed mutagenesis of regions of the tcpA gene that encode pilin domains implicated by immunological analysis with monoclonal antibodies to directly mediate colonization. Other studies involving tcpA, itself, include determining the sequence of the tcpA gene of El Tor biotype isolates from the recent Peru epidemic so that knowledge from ongoing work with classical strains can be extended to these strains of current interest. In addition to further characterizing tcpA, sequence analysis of the rest of the tcp gene cluster with identification of corresponding gene products by in vivo T7 expression of appropriate subclones will be completed. The products visualized by T7, or immunologically utilizing antibodies directed against corresponding synthetic peptides, will be localized. This should finally allow conclusion as to whether any additional adhesin proteins are involved in the TCP structure. Two genes involved in TCP biogenesis have already been characterized to the point of identifying them as proteins that help define new classes of biological function. One, TcpJ, is a leader peptidase specific for the for the TcpA leader peptide and export. The other, TcpG, is a new periplasmic thiol:disulfide isomerase required not only for TCP function, but for toxin secretion. The details by which these proteins function will be addressed by mutagenesis and allele specific suppressor analysis. Lastly, additional putative colonization factors elaborated by El Tor biotype strains will be studied. Initially, the sequence of a Tn5 insertion mutation that negates mannose-sensitive hemagglutination, and that has recently been cloned in our laboratory, will be determined. This will allow localization and functional studies of the putative adherence molecule. The contribution of this hemagglutinin for colonization, as compared to the TCP contribution for isogenic El Tor strains, will be assessed by determining competitive indexes in the infant mouse cholera model.